Anti-atherogenic mechanisms of high density lipoprotein: Effects on myeloid cells

In some settings increasing high density lipoprotein (HDL) levels has been associated with a reduction in experimental atherosclerosis. This has been most clearly seen in apolipoprotein A-I (apoA-I) transgenic mice or in animals infused with HDL or its apolipoproteins. A major mechanism by which these treatments are thought to delay progression or cause regression of atherosclerosis is by promoting efflux of cholesterol from macrophage foam cells. In addition, HDL has been described as having anti-inflammatory and other beneficial effects. Some recent research has linked anti-inflammatory effects to cholesterol efflux pathways but likely multiple mechanisms are involved. Macrophage cholesterol efflux may have a role in facilitating emigration of macrophages from lesions during regression. While macrophages can mediate cholesterol efflux by several pathways, studies in knockout mice or cells point to the importance of active efflux mediated by ATP binding cassette transporter (ABC) A1 and G1. In addition to traditional roles in macrophages, these transporters have been implicated in the control of hematopoietic stem cell proliferation, monocytosis and neutrophilia, as well as activation of monocytes and neutrophils. Thus, HDL and cholesterol efflux pathways may have important anti-atherogenic effects at all stages of the myeloid cell/monocyte/dendritic cell/macrophage lifecycle. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).     

Scavenger receptor for malondialdehyde-modified high density lipoprotein on rat sinusoidal liver cells

We report here the presence of a membrane-associated receptor which mediates endocytic uptake of malondialdehyde-modified high density lipoprotein (MDA-HDL) on sinusoidal liver cells. Binding of [125I]MDA-HDL to the cells was followed by internalization and degradation in lysosomes. The binding and lysosomal degradation of [125I]MDA-HDL were effectively inhibited by unlabeled MDA-HDL and acetyl-HDL. However, formaldehyde-treated serum albumin or low density lipoprotein modified either by acetylation or malondialdehyde, ligands known to undergo receptor-mediated endocytosis by sinusoidal liver cells, did not affect the binding of [125I]MDA-HDL to the cells. These results indicate that a receptor for MDA-HDL is described as a distinct member among the scavenger receptors for chemically modified proteins.     

Biosynthesis of high density lipoprotein by chicken liver: nature of nascent intracellular high density lipoprotein

Young chickens were administered L-[(3)H]leucine and after 10 or 30 min the livers were removed and fractioned into rough (RER) and smooth (SER) endoplasmic reticulum fractions and into light, intermediate, and heavy golgo cell fractions. The labeled high density lipoprotein (HDL), contained within these intracellular organelles was isolated either by immunoprecipitation using rabbit antiserum to rooster HDL, or by ultracentrifugal glotation between densities 1.063 and 1.21 g/ml. The radioactive apoproteins of nascent HDL were analyzed by SDS PAGE and detected by fluorography. Analyses of radioactive apoproteins obtained by immunoprecipitation from the contents of the RER, the SER, and the three golgi complex fractions revealed only one apoprotein, A1. The C peptide present in serum HDL was not detected intracellularly. The radioactive apoprotein A1 which is present within the cisternae of the RER and the SER fractions failed to float, whereas apoprotein A1, present within the golgi apparatus, readily floated between densities 1.063 and 1.21 g/ml. The HDL particles, isolated by flotation from the golgi apparatus content, were further characterized by lipid and protein analyses and by electron microscopy. Golgi HDL particles have the same density as serum HDL. On a percentage basis, golgi HDL contains less protein and more phospholipids than does serum HDL. Morphologically, golgi HDL is different in appearance from serum HDL. It is more heterogeneous in size, with most of the particles ranging 8.3-25 nm in diameter. The spherical particles contain small membrane tails. Occasionally, a few disk-shaped bilayer structures are also found within the golgi apparatus. These studies show that the newly synthesized apoprotein A1, present within the RER and the SER cell fractions, is not fully complexed with lipid and that apoprotein A1 does not acquire sufficient lipid to float at the proper HDL density until it enters the golgi apparatus. The difference in chemical composition and the heterogeneous size of golgi HDL may be attributed to the different stages of HDL maturation.     

Effect of homocysteinylation on high density lipoprotein physico-chemical properties

High density lipoproteins (HDL) exert a protective effect against homocysteinylation due to the activity of the enzyme paraoxonase/thiolactonase associated to the lipoprotein surface. However, a small amount of N-homocysteinylated HDL (N-Hcy-HDL) is present in human plasma, suggesting that homocysteinylation of plasma lipoproteins occurs in vivo.Aim of the present study was to investigate the effect of homocysteinylation on apoprotein structure and physico-chemical properties of HDL using the analysis of the fluorescent emission spectra of tryptophan and Laurdan (6-dodecanoyl-2-dimethyl-aminonaphthalene). Our results demonstrated that the increase in –SH groups in HDL homocysteinylated in vitro (Hcy-HDL) was associated with apoprotein conformational changes and modifications of physico-chemical properties.A significant decrease of paraoxonase and lactonase activity of HDL bound PON1 has also been observed in Hcy-HDL. A significant decrease of the enzyme activity has been observed also in purified PON1 homocysteinylated following the same experimental conditions used for HDL. Moreover, we demonstrated that oxidized HDL were more susceptible to homocysteinylation with respect to control HDL.The modifications of apoprotein conformation and physico-chemical properties in Hcy-HDL and the decrease of paraoxonase-1 activity could affect the protective effect of HDL against oxidative damage and/or homocysteinylation and could contribute to accelerated atherosclerosis in patients affected by diseases associated with oxidative damage, in renal disorders and in patients affected by genetic or nutritional disorders of homocysteine or folate metabolism.     

Lipid monolayers: interactions with the apoprotein of high density plasma lipoprotein

Monolayer techniques were used to study the interactions of various lipids (cholesterol, lysophosphatidyl choline, phosphatidal ethanolamine, phosphatidyl choline, sphingomyelin, stearic acid, and lipids extracted from plasma high density lipoproteins and very low density lipoprotein) with the lipid-free protein subunit of rat plasma high density lipoprotein and with rat plasma albumin. The proteins were injected under the lipid monolayer at fixed area, and the increase in surface pressure (decrease in surface tension) was measured as a function of time. With all lipids, both the rate and magnitude of this increase were greater with the apolipoprotein than with albumin. The degree of film penetration of pure lipid films (at an initial film pressure of 15 dynes/cm) by the two proteins followed the same order: cholesterol > phosphatidal ethanolamine > phosphatidyl choline > stearic acid > sphingomyelin > lysophosphatidyl choline. Other variables studied were protein concentration, initial film pressure, and pH. Two distinctive properties of the apolipoprotein were the penetration of lipid films at pressures above the collapse pressure of the protein, and the formation of a film even at low salt concentration. High surface activity and strong interaction of HDL-protein with lipid monolayers may be associated with the flexibility of the protein molecule due to absence of disulfide bridges. The unusual surface activity of HDL-protein may be intimately related to the mechanism of formation of the lipoprotein.     

Small high density lipoprotein subclasses: some of their physico-chemical properties and stability in solution

Small high density lipoproteins (SHDL) contribute to the protection from atherosclerosis, but detailed information about their properties is not available yet. We isolated four of the smallest HDL subclasses that contain apoA-I alone, the small lipoprotein A-I (SLpAI), by their separation on gradient polyacrylamide gel followed by electroelution. Their physico-chemical properties were calculated from their displacement in non-denaturing gradient polyacrylamide gel under the effect of electrical potential. The properties are: Stokes' radii 2.96-3.56 nm; molecular masses 42-70 kDa; net negative charge 7.2-13.5; surface charge densities 3139-4069 -esu.cm(-2); surface potentials 10.6-15.7 -mV; coefficients of friction 5.74-6.90 x 10(-8) g.s(-1); and diffusion coefficients 5.76-6.94 7times; 10(-7) cm(2).s(-1). We found that these particles were of low stability as they underwent molecular transformation into larger particles on storage. The estimated dimensions of these particles do not support ellipsoidal shape, therefore, the most probable shape is spherical; consequently, their hydrated characteristics were estimated. We conclude that these particles have high values of negative surface charge and diffusion coefficients, and are of low stability. Their small Stokes' radii were similar to each other and they are spherical and highly hydrated.     

High density lipoprotein and low density lipoprotein catabolism by human liver and parenchymal and non-parenchymal cells from rat liver.

The capacity of the homogenates from human liver, rat parenchymal cells, rat non-parenchymal cells and total rat liver for the breakdown of human and rat high density lipoprotein (HDL) and human low density lipoprotein (LDL) was determined. Human HDL was catabolized by human liver, in contrast to human LDL, the protein degradation of which was low or absent. Human and rat HDL were catabolized by both the rat parenchymal and non-parenchymal cell homogenates with, on protein base, a 10-times higher activity in the non-parenchymal liver cells. This implies that more than 50% of the total liver capacity for HDL protein degradation is localized in these cell types. Human LDL degradation in the rat could only be detected in the non-parenchymal cell homogenates. These findings are discussed in view of the function of HDL and LDL as carriers for cholesterol.     

Dynamic properties of human high density lipoprotein apoproteins.

This study was designed to identify a method for the measurement of human high density lipoprotein subfraction (HDL2 and HDL3) metabolism. Apolipoproteins A-I, A-II, and C, the major HDL apoproteins, were radioiodinated and incorporated individually into HDL2 and HDL3 in vitro. Using a double label technique, the turnover of apoA-I in HDL2 and HDL3 was measured simultaneously in a normal male. The apoprotein exchanged rapidly between the two subfractions, evidenced by equilibration of their apoA-I specific activity. Radiolabeled apoA-II, incorporated into the subfractions, showed a similar exchange in vitro. Incubation of 131I-labeled very low density lipoproteins (VLDL) with HDL or its subfractions resulted in transfer of C proteins from VLDL to the HDL moiety. The extent of transfer was dependent on the HDL subfraction present; 50% of the VLDL apoC was transferred to HDL3, while the transfer to total HDL and HDL2 was 69% and 78%, respectively. ApoC also exchanged between HDL2 and HDL3, again showing a preference for the former and suggesting a primary metabolic relationship between VLDL and HDL2. Overall, the study indicates that apoA-I, apoA-II, and the C proteins exist in equilibrium between HDL2 and HDL3. This phenomenon precludes their use as probes for HDL subfraction metabolism in humans.     

Effects of cimetidine and ranitidine on high density lipoprotein cholesterol concentrations.

To assess the effect of cimetidine and ranitidine on high density lipoprotein (HDL) cholesterol concentration two groups of eight patients with duodenal ulcer or oesophagitis matched for age, sex, and cigarette consumption were given either cimetidine 1 g daily or ranitidine 300 mg daily for one month. There was no significant change in the cholesterol content of HDL and its subfraction HDL3 after treatment with ranitidine or cimetidine, or in the cholesterol content of the subfraction HDL2 after treatment with ranitidine; the HDL2 cholesterol concentration was, however, significantly increased after treatment with cimetidine. Further studies are being undertaken to establish the mechanism of this effect.     

Glycosaminoglycan—lipoprotein interactions: 2. Structure of heparin-releted variants with high affinity for low density lipoprotein

A particular heparan sulphate fraction which possessed the largest proportion of high affinity variants for human low density lipoprotein contained almost equal proportions of the repeating units l-iduronosyl(O-sulphate)N-sulphamidoglucosamine and d-glucoronosyl-N-acetylglucosamine. The heparan sulphate was fractionated on lipoprotein-agarose into three populations. Results of periodate oxidation—alkaline elimination indicated that the size of the completely N-sulphated block regions increased with increasing affinity. In contrast, the number of consecutive l-iduronosyl(O-sulphate)-containing repeats decreased with increasing affinity towards lipoprotein. After selective periodate oxidation—alkaline scission of d-glucoronic acid residues only a portion of the heparan sulphate fragments retained high affinity for lipoprotein. This portion consisted of fragments larger than dodecasaccharide which contained both l-iduronic acid-O-sulphate and non-sulphated uronic acid residues (−) 2:1). No affinity or little affinity was displayed by fragments (of comparable size) that contained only sulphated l-iduronic acid residues.     

In vivo uptake of human and rat low density and high density lipoprotein by parenchymal and nonparenchymal cells from rat liver.

The relative contribution of the parenchymal and nonparenchymal rat liver cells to the hepatic uptake of human and rat high density lipoprotein (HDL) and low density lipoprotein (LDL) was determined in vivo. Nonparenchymal cells, isolated 6 h after intravenous injection of iodinated human HDL and LDL, contained respectively 4.2 and 6.3 times the amount of trichloroacetic acid-precipitable radioactivity per mg cell protein as compared to parenchymal cells. For rat iodinated HDL and LDL these factors were 3.4 and 4.1, respectively. These results indicate that nonparenchymal liver cells play a substantial role in the hepatic uptake of human and rat HDL and LDL in vivo.     

Effects of fat ingestion on high density lipoprotein profiles in human sera

Serum concentrations of triacylglycerol, apolipoprotein A-I, apolipoprotein A-II, HDL2, and HDL3 were determined in sera of nine normolipidemic adult males, just before and 3, 5, and 8 hr after ingestion of 250 ml of cream (100 g of triacylglycerol). In all individuals a rapid hypertriglyceridemic response was observed. Triacylglycerol concentrations increased from 624 +/- 124 mg/liter of serum to 1435 +/- 350 mg/liter of serum 3 hr after cream ingestion. In most individuals the hypertriglyceridemic response was followed by a decline in serum triacylglycerol concentration to below basic levels. As a result of cream ingestion, small but statistically highly significant increases in serum cholesterol and apolipoprotein A-I concentrations were observed that persisted till the end of the observation period. In most individuals a small rise in the apolipoprotein A-II concentration in serum was also present. Marked changes were observed in serum HDL as illustrated in the HDL absorption at 280 nm and cholesterol profiles obtained by single-spin rate-zonal density gradient ultracentrifugation of the sera. Due to a prominent increase in phospholipids (up to about 18%) and a smaller increase in protein (up to about 6%), flotation rates and concentrations of HDL2 as well as HDL3 increased. These changes in HDL subclass flotation characteristics and chemical composition are best explained by uptake of surface material from chylomicrons by existing HDL2 and HDL3 particles. The data do not support a previously proposed concept in which HDL3 is converted into HDL2 by uptake of surface remnants formed during catabolism of triglyceride-rich lipoproteins.     

Distinction in the mode of receptor-mediated endocytosis between high density lipoprotein and acetylated high density lipoprotein: evidence for high density lipoprotein receptor-mediated cholesterol transfer

The interactions of high density lipoprotein (HDL) and acetylated high density lipoprotein (acetyl-HDL) with isolated rat sinusoidal liver cells have been investigated. Cellular binding of 125I-acetyl-HDL at 0 degrees C demonstrated the presence of a specific, saturable membrane-associated receptor. This receptor was affected neither by formaldehyde-treated albumin nor by low density lipoprotein modified either by acetylation or malondialdehyde, ligands known to undergo receptor-mediated endocytosis by the cells, indicating that the receptor for acetyl-HDL constitutes a distinct class among the scavenger receptors for chemically modified proteins. Parallel binding experiments using 125I-HDL also revealed the presence on these cells of a receptor for unmodified HDL. The ligand specificities of these two receptors were similar to each other except that the acetyl-HDL receptor was sensitive to polyanions such as dextran sulfate and fucoidin. Interaction of HDL with the cells at 37 degrees C was totally different from that of acetyl-HDL. Cellular binding of HDL was not accompanied by subsequent intracellular degradation of its apoprotein moiety, whereas its cholesterol moiety was significantly transferred to the cells. In contrast, acetyl-HDL was endocytosed and underwent lysosomal degradation as a holoparticle. This shift in receptor-recognition from the HDL receptor to the acetyl-HDL receptor was accomplished by acetylation of approximately 8% of the total lysine residues of HDL apoprotein. This unique difference in endocytic behavior between HDL and acetyl-HDL suggests a potential link of the HDL receptor to HDL-mediated cholesterol transfer in sinusoidal liver cells.     

Characterization of nascent high density lipoprotein subfractions from perfusates of rat liver

Nascent high density lipoprotein (HDL) (1.063 less than d less than 1.21 g/ml) was isolated from recirculating rat liver perfusates and separated by heparin-Sepharose chromatography into a nonretained fraction (NR) and a fraction (R) that eluted with 0.5 M NaCl. Fractions NR and R contained 70% and 30% of the nascent HDL protein, respectively. ApoB-containing particles were removed from fraction R by chromatography on concanavalin A. The protein composition of fractions NR and R was 40% and 29%, respectively. Fraction NR contained 25% apoA-I, 11% apoA-IV, 24% apoE, and 38% apoC. Fraction R contained primarily apoE (81% of total protein). The lipid composition of NR and R, respectively, was: triglyceride 44% and 26%, phospholipid 41% and 57%, cholesterol 8% and 13%, and cholesteryl ester 7% and 4%. Fractions NR and R had molecular weights of 400,000 and 860,000, respectively, as calculated from the Stokes radius. Negative staining electron microscopy indicated that both fractions consisted mainly of spherical particles (260-280 A) but some stacked disks were seen in fraction R. Livers perfused by the single-pass technique produced fractions NR and R in the same ratio as livers perfused by recirculation. The apolipoprotein compositions were similar to those in the recirculating perfusion; however, both fractions NR and R had more triglyceride (greater than 50% of total lipid). An HDL fraction was also isolated from liver perfusates by a combination of molecular sieve and heparin-Sepharose affinity chromatography. This HDL contained triglyceride but no apoB, indicating that triglyceride-rich HDL particles are not an artifact of ultracentrifugation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for two sites on rat liver plasma membranes which interact with high density lipoprotein.

There is little dispute that high density lipoprotein (HDL) binds to cells, however, the nature of the interaction is not fully understood. We now present evidence for a new binding site of higher affinity but lower capacity than the sites previously described in the literature. This new site is characterized by high affinity/low capacity for HDL binding (Kd = 0.94 microgram/ml, Bmax = 36 ng/mg), while the low affinity site (Kd = 36 micrograms/ml, Bmax approximately 700 ng/mg) appears to be consistent with the literature values for the interaction of HDL with cells and isolated membranes. Proteolysis of HDL with trypsin abolished its interaction with the high affinity site, suggesting an apolipoprotein requirement, while having no effect on binding to the lower affinity site. Kinetic rates of association/dissociation were determined in order to further characterize the high affinity site. At a concentration which favored the binding of HDL with the high affinity site (1 microgram/ml, 37 degrees C), the time course of association of HDL with rat liver plasma membranes, displayed a biphasic pattern, requiring 6-8 h to reach the level of binding predicted from the saturation studies. The second phase was highly sensitive to temperature, being considerably slower at 24 degrees C and totally abolished at 0 degrees C. A kinetic Kd, derived from the measured association and dissociation rate constants (Kd = 0.31 microgram/ml), was found to be of a similar magnitude to the Kd calculated for the high affinity site by Scatchard analysis (Kd = 0.94 microgram/ml). In summary, the high affinity site on rat liver plasma membranes displays an apoprotein requirement and kinetic parameters, consistent with a ligand-receptor interaction.